Location specific small RNA annealing to the HCV 5’ UTR promotes Hepatitis C Virus replication by favoring IRES formation and stimulating virus translation

ABSTRACT Hepatitis C virus (HCV) genome replication requires annealing of a liver specific small-RNA, miR-122 to 2 sites on 5’ untranslated region (UTR). Annealing has been reported to a) stabilize the genome, b) promote translation, and c) induce the canonical HCV 5’ UTR Internal Ribosome Entry Site (IRES) structure. In this report we identify the relative impact of small RNA annealing on the three functions ascribed to miR-122 and generate a mechanistic model for miR-122 promotion of HCV. First, we identified that perfectly complementary small RNAs that anneal to different locations on the HCV 5’ UTR stimulate replication with varying efficiencies and mapped the region on the HCV genome to which small RNA annealing promotes virus replication. Second, by using a panel of small RNAs that promote with varying efficiencies we link HCV replication induction with translation stimulation and 5’ UTR RNA structure modifications. However, replication promotion was not linked to genome stabilization since all small RNAs tested could stabilize the viral genome regardless of their ability to promote replication. Thus, we propose that miR-122 annealing promotes HCV replication primarily by activating the HCV IRES and stimulating translation, and that miR-122-induced HCV genome stabilization is insufficient alone but enhances virus replication. Graphical Abstract